Gregory T. Roman

Improved Temporal Resolution for in Vivo Microdialysis by Using Segmented Flow

Microdialysis sampling probes were interfaced to a segmented flow system to improve temporal resolution for monitoring concentration dynamics. Aqueous dialysate was segmented into nanoliter plugs by pumping sample stream into the base of a tee channel structure microfabricated on a PDMS chip that had an immiscible carrier phase (perfluorodecalin) pumped into the cross arm of the tee. Varying the oil flow rate from 0.22 to 6.3 microL/min and sample flow rate from 42 to 328 nL/min allowed control of plug volume, interval between plugs, and frequency of plug generation between 6 and 28 nL, 0.6 and 10 s, and 0.1 and 1.7 Hz, respectively. Temporal resolution of the system, determined by measuring fluorescence in individual sample plugs following step changes of fluorescein concentration at the sampling probe surface, was as good as 15 s. Temporal resolution was independent of both sampling flow rate and distance that samples were pumped from the sampling probe. This effect is due to the prevention of Taylor dispersion of the sample as it was transported by segmented flow. In contrast, without flow segmentation, temporal resolution was worsened from 25 to 160 s as the detection point was moved from the sampling probe to 40 cm downstream. Glucose was detected by modifying the chip to allow enzyme assay reagents to be mixed with dialysate as sample plugs formed. The resulting assay had a detection limit of 50 microM and a linear range of 0.2-2 mM. This system was used to measure glucose in the brain of anesthetized rats. Basal concentration was 1.5 +/- 0.1 mM (n = 3) and was decreased 60% by infusion of high-K(+) solution through the probe. These results demonstrate the potential of microdialysis with segmented flow to be used for in vivo monitoring experiments with high temporal resolution.

Microfluidic Chip for High Efficiency Electrophoretic Analysis of Segmented Flow from a Microdialysis Probe and in Vivo Chemical Monitoring

An effective method for in vivo chemical monitoring is to couple sampling probes, such as microdialysis, to online analytical methods. A limitation of this approach is that in vivo chemical dynamics may be distorted by flow and diffusion broadening during transfer from sampling probe to analytical system. Converting a homogeneous sample stream to segmented flow can prevent such broadening. We have developed a system for coupling segmented microdialysis flow with chip-based electrophoresis. In this system, the dialysis probe is integrated with a PDMS chip that merges dialysate with fluorogenic reagent and segments the flow into 8-10 nL plugs at 0.3-0.5 Hz separated by perfluorodecalin. The plugs flow to a glass chip where they are extracted to an aqueous stream and analyzed by electrophoresis with fluorescence detection. The novel extraction system connects the segmented flow to an electrophoresis sampling channel by a shallow and hydrophilic extraction bridge that removes the entire aqueous droplet from the oil stream. With this approach, temporal resolution was 35 s and independent of distance between sampling and analysis. Electrophoretic analysis produced separation with 223,000 +/- 21,000 theoretical plates, 4.4% RSD in peak height, and detection limits of 90-180 nM for six amino acids. This performance was made possible by three key elements: (1) reliable transfer of plug flow to a glass chip; (2) efficient extraction of aqueous plugs from segmented flow; (3) electrophoretic injection suitable for high efficiency separation with minimal dilution of sample. The system was used to detect rapid concentration changes evoked by infusing glutamate uptake inhibitor into the striatum of anesthetized rats. These results demonstrate the potential of incorporating segmented flow into separations-based sensing schemes for studying chemical dynamics in vivo with improved temporal resolution.

High efficiency micellar electrokinetic chromatography of hydrophobic analytes on poly(dimethylsiloxane) microchips

This paper describes a simple method for the effective and rapid separation of hydrophobic molecules on polydimethylsiloxane (PDMS) microfluidic devices using Micellar Electrokinetic Chromatography (MEKC). For these separations the addition of sodium dodecyl sulfate (SDS) served two critical roles - it provided a dynamic coating on the channel wall surfaces and formed a pseudo-stationary chromatographic phase. The SDS coating generated an EOF of 7.1 x 10(-4) cm(2) V(-1) s(-1) (1.6% relative standard deviation (RSD), n = 5), and eliminated the absorption of Rhodamine B into the bulk PDMS. High efficiency separations of Rhodamine B, TAMRA (6-carboxytetramethylrhodamine, succinimidyl ester) labeled amino acids (AA), BODIPY FL CASE (N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)cysteic acid, succinimidyl ester) labeled AAs, and AlexaFluor 488 labeled Escherichia coli bacterial homogenates on PDMS chips were performed using this method. Separations of Rhodamine B and TAMRA labeled AAs using 25 mM SDS, 20% acetonitrile, and 10 mM sodium tetraborate generated efficiencies > 100,000 plates (N) or 3.3 x 10(6) N m(-1) in <25 s with run-to-run migration time reproducibilities <1% RSD over 3 h. Microchips with 30 cm long serpentine separation channels were used to separate 17 BODIPY FL CASE labeled AAs yielding efficiencies of up to 837,000 plates or 3.0 x 10(6) N m(-1). Homogenates of E. coli yielded approximately 30 resolved peaks with separation efficiencies of up to 600,000 plates or 2.4 x 10(6) N m(-1) and run-to-run migration time reproducibilities of <1% RSD over 3 h.

Electrokinetic trapping using titania nanoporous membranes fabricated using sol-gel chemistry on microfluidic devices.

We have developed a new method for analyte preconcentration on a microfluidic device using a porous membrane fabricated via sol–gel chemistry. These porous membranes were fabricated within the channels of glass microfluidic devices exploiting laminar flow to bring an alcoholic sol–gel precursor (titanium isopropoxide in 2‐propanol) into contact with an alcohol–water solution at a channel cross intersection. These two streams reacted at the fluidic interface to form a porous titania membrane. The thickness of the membrane could be altered by changing the [H2O]. Analyte concentration was accomplished by applying a voltage across the titania membrane. The level of analyte enrichment was monitored, and enrichment factors of above 4000 in 400 s were obtained for 2,7‐dichlorofluorescein.

Synthesis and Characterization of a Poly(dimethylsiloxane)-Poly(ethylene oxide) Block Copolymer for Fabrication of Amphiphilic Surfaces on Microfluidic Devices

A poly(dimethylsiloxane)-poly(ethylene oxide) (PDMS-PEO) vinyl terminated block copolymer has been synthesized via a simple hydrosilylation reaction between hydride-terminated PDMS and PEO divinyl ether. This prepolymer can be subsequently cross-linked into an elastomer in a second hydrosilylation reaction involving a methylhydrosiloxane-dimethylsiloxane copolymer, forming a material suitable for the purposes of fabricating microfluidic devices. The presence of the PEO block in the prepolymer chain results in a much more hydrophilic material following cross-linking. The surface water contact angle of the PDMS-PEO material is 65 degrees +/- 3 (n = 6), as opposed to approximately 110 degrees for native PDMS. Droplets of water straddled by air within molded channels of the PDMS-PEO are concave in shape with contact angles where the fluid meets the side walls of 32 degrees +/- 4 (n = 8), while droplets in PDMS microchannels are more convex with contact angles of 95 degrees +/- 6 (n = 6). The length of the PDMS-PEO prepolymer chain and the multifunctional hydride cross-linker chains appear to dictate the durability of the elastomeric material. Youngs modulus measurements yielded values of 0.94 +/- 0.08, 2.6 +/- 0.8, and 1.91 +/- 0.06 MPa for a [5% vinyl excess prepolymer and 10-fold excess of cross-linker], [10% vinyl excess prepolymer and 5-fold excess of cross-linker], and 10:1 PDMS, respectively, confirming that the elasticity of the cross-linked PDMS-PEO is similar to that of PDMS (Sylgard 184:10:1 mixture of elastomeric base to elastomer curing agent). The PDMS-PEO material still possesses enough PDMS character to allow molded channel architectures to be sealed between two pieces of the block copolymer by conformal contact. As a result of the more hydrophilic nature of the material, the channels of devices fabricated from this polymer are self-filling when using aqueous buffers, making it more user-friendly than PDMS for applications calling for background electrolytes void of organic modifiers. Different compositions of PDMS-PEO devices feature different electroosmotic flow values with the 5% vinyl excess prepolymer EOF values of 2.5 +/- 0.7 x 10(-4) and 5.7 +/- 0.8 x 10(-4) cm(2)/(V s) at pHs 6 and 9, respectively, and 1.2 +/- 0.3 x 10(-4) and 2.5 +/- 0.3 x 10(-4) cm(2)/(V s) for the 10% vinyl excess prepolymer device at pHs 6 and 9, respectively.

Optical microscopy studies of polymer/liquid-crystal diffractive optics

Electrically switchable diffraction gratings having periods as small as 5 μm and incorporating nematic LC confined within channels formed in poly(vinyl alcohol) (PVA) films were prepared and characterized. Gratings were produced by first using conventional photolithographic procedures to prepare reusable surface-relief grating molds from a common photoresist (SU-8). An aqueous PVA solution was then drop coated onto the mold and dried, The PVA film was subsequently peeled from the mold and pressed (channel side down) onto an indium tin oxide (ITO) coated glass slide. The channels were then filled with LC using capillary action. Finally, a second ITO-coated slide was pressed onto the backside of the PVA film, forming a polymer/LC grating cell. The diffraction efficiency of each grating was measured as a function of applied electric field strength using 488 nm light. Beam diffraction was greatest in the absence of the field and fell to zero for applied fields of less than 10 V/μm. These studies and atomic force microscopy results showed the LC channels to be ≈ 50-100 nm deep. Multiphoton excited fluorescence microscopy (MPEFM) was used to show the LC was oriented predominantly parallel to the long axis of the channels in the zero field state. Significant nonuniformity observed in the LC orientation was attributed to channel (PVA) wall roughness. Time-resolved MPEFM was used to monitor the LC reorientation process on submicron length scales. The local LC reorientation dynamics were also strongly perturbed by channel wall roughness.